Method for rapidly detecting the total mercury content of soil in urban relocation sites by using archaea molecular marker otu69

ABSTRACT

The present invention discloses a method for rapidly detecting the total mercury content of soil in urban relocation sites using archaea molecular marker OTU69. The present invention provides a DNA molecule (probe), as shown in SEQ ID NO.1 of the sequence listing. The present invention also protects the application of the probe in detecting or assisting in detecting the total mercury content of the soil. The present invention also protects the application of the probe in comparing the total mercury content of the soil in different plots. Using the method provided by the present invention to detect the total mercury content of the soil or compare the total mercury content of the soil of different plots has the following advantages: small sample demand, no need for pre-treatment, short required time, low labor cost, and realizing the rapid automatic detection of large quantities of samples.

TECHNICAL FIELD

The present invention belongs to the field of biotechnology and relatesto a method for rapidly detecting the total mercury content of soil inurban relocation sites using archaea molecular marker OTU69.

BACKGROUND

With the rapid development of urbanization, cities, especiallymegacities, have entered a stage of urban development dominated by“urban redevelopment”. A large number of urban relocation sites haveappeared, especially in cities in economically developed areas along theeastern coast. Taking Shanghai as an example, 80%-90% of the green landin the built-up area was developed on the demolition of urban villagesand the relocation of old factories. Scientific and objective monitoringand evaluation for its soil quality is a prerequisite and an importantreference basis for ecological restoration and landscaping in urbanrelocation sites.

At present, the detection of soil quality indicators in urban relocationsites mainly follows traditional physical and chemical detectionmethods, which require a lot of samples, a complicated pretreatmentprocess, a long cycle, and a high cost on human resources. So it isdifficult to achieve rapid detection of large quantities of samples.Soil microorganisms have comprehensive, sensitive, and functionalcharacteristics to the changes in the soil environment. It is of greatpractical significance to explore the rapid and automatic detectiontechnology for soil quality indicators in urban relocation sites bydetecting the abundance of specific microbial groups.

Element mercury is one of the most harmful heavy metals to the humanbody and is one of the 129 priority control pollutants. The mercury inthe soil mainly comes from 1) The soil parent material, the mercury inthe soil parent material is the basic source in the soil. The content ofmercury in the primary rock directly determines the content of mercuryin the soil. 2) Atmospheric deposition, after the mercury in theatmosphere enters the soil, most of it is quickly absorbed or fixed bythe clay minerals and organic matter in the soil and enriched in thesurface of the soil. 3) Direct pollution, mainly including the stackingof industrial production waste and urban domestic garbage, unreasonableapplication of mercury-containing fertilizers and pesticides,irrigation, etc. Mercury and its compounds have strong neurotoxicity andteratogenic effects and pose a serious threat to the ecologicalenvironment and human health. The problem of soil mercury pollution hasgradually received attention.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for rapidlydetecting the total mercury content of the soil in urban relocationsites by using archaea molecular marker OTU69.

The present invention also provides a method (Method A) for detectingtotal mercury content in the soil, which includes the following steps:

Using the total DNA of the soil sample as a template to performreal-time fluorescent quantitative PCR; The amplification primer pair ofreal-time fluorescence quantitative PCR is composed of primer524F-10-ext shown as SEQ ID NO.4 of the sequence listing and primerArch958R-mod shown as SEQ ID NO.5 of the sequence listing. Thenucleotide sequence of the probe for real-time fluorescence quantitativePCR is shown as SEQ ID NO.1 of the sequence listing.

The Ct value is obtained by real-time fluorescent quantitative PCR; thecopy number is calculated according to the Ct value, and the totalmercury content in the soil sample is calculated by the copy numbercontent in the soil sample.

The probe of real-time fluorescent quantitative PCR is Taqman probe. TheTaqman probe has a fluorescent group at the 5′end and a fluorescencequenching group at the 3′end. The fluorescent group may specifically beFAM. The fluorescence quenching group may specifically be TAMRA.

The copy number is the copy number of the target fragment of the probe.

The method for calculating the total mercury content in the soil sampleby calculating the copy number content in the soil sample is:substituting the copy number content of the soil sample into a linearequation to obtain the total mercury content of the soil sample.

The linear equation is y=−0.1811x+0.6508; y represents total mercurycontent, and the unit is mg/kg; x represents copy number content, andthe unit is ×10⁷ copies/g. R²=0.959 for the linear equation.

The method of calculating the copy number based on the Ct value is:Substituting the Ct value into the standard curve equation to obtain thecopy number.

The preparation method of the standard curve equation is: ligate the DNAmolecule shown as SEQ ID NO.2 of the sequence listing to the pMD18-Tvector to obtain OTU69 standard plasmid; and use the OTU69 standardplasmid to produce a standard curve equation with the logarithm of thecopy number as the independent variable and Ct as the dependentvariable. The logarithm of the copy number is the logarithm with base 10of the copy number.

Preparation method of total DNA of soil samples: AdoptMoBioPowerSoil®DNA Isolation Kit (MoBio Laboratories, Carlsbad, Inc., CA, USA) toextract total DNA of soil samples.

The real-time fluorescent quantitative PCR was performed onaLightCycler® 96 real-time fluorescent quantitative PCR instrument.

The real-time fluorescent quantitative PCR reaction system (20 μL) is:10 μL Premix Ex Taq (Takara, Dalian, China), 0.4 μL primer 524F-10-ext,0.4 μL primer Arch958R-mod, 0.2 μL probe, 2 μL template solution and 7μL sterile water. In the reaction system, the concentration of primer524F-10-ext is 0.2 μM, wherein the concentration of primer Arch958R-modis 0.2 μM, and the concentration of probe is 0.1 μM. In the reactionsystem, the content of template DNA is Ing.

The real-time fluorescent quantitative PCR reaction program:pre-denaturation at 95° C. for 120 s, denaturation at 95° C. for 10 s,annealing extension at 60° C. for 45 s, 45 cycles.

The present invention also provides a method for comparing the totalmercury content of the soil in different plots (Method B), whichincludes the following steps:

testing the soil samples of more than two plots according to Method A;

comparing the total mercury content in the soil of each plot based onthe test results.

The present invention provides a DNA molecule (probe), shown in SEQ IDNO.1 of the sequence listing.

The DNA molecule (probe) may or may not be labeled with a label. Thelabel refers to any atom or molecule that can be used to provide adetectable effect and can be attached to a nucleic acid. The labelincludes but not limited to dyes; radiolabels, such as 32P; bindingmoieties, such as biotin; haptens, such as digoxin (DIG); luminescent,phosphorescent, or fluorescent moieties; and fluorescent dyes alone orfluorescent dyes combined with moieties, of which the emission spectrumcan be inhibited or shifted by fluorescence resonance energy transfer(FRET). The labels can provide a signal that can be detected byfluorescence, radioactivity, colorimetry, gravimetry, X-ray diffractionor absorption, magnetism, enzyme activity, and so on. Labels can be acharged moiety (positive or negative), or optionally can be neutral.Labels may include a nucleic acid or protein sequence or a combinationthereof, as long as the sequence containing labels is detectable. Insome embodiments, directly detect the nucleic acid without the label(e.g., read the sequence directly).

The present invention provides a Taqman probe, its nucleotide sequenceshown as SEQ ID NO.1 of the sequence listing. The Taqman probe has afluorescent group at the 5′end and a fluorescence quenching group at the3′end. The fluorescent group may specifically be FAM. The fluorescencequenching group may specifically be TAMRA.

The present invention also claims the application of the DNA molecule orthe Taqman probe in detecting or assisting in detecting the totalmercury content of the soil.

The present invention also claims the application of the DNA molecule orthe Taqman probe in comparing the total mercury content of the soil indifferent plots.

The present invention also claims the primer-probe set, which iscomposed of a specific primers pair and a specific probe; The specificprimers pair is composed of the primer 524F-10-ext shown in SEQ ID NO.4of the sequence listing and the primer Arch958R-mod shown in SEQ ID NO.5of the sequence listing; The nucleotide sequence of the specific probeis shown in SEQ ID NO.1 of the sequence listing. The specific probe is aTaqman probe. The Taqman probe has a fluorescent group at its 5′end anda fluorescence quenching group at its 3′end. The fluorescent group mayspecifically be FAM. The fluorescence quenching group may specificallybe TAMRA.

The present invention also protects the application of the primer-probeset in detecting or assisting in detecting the total mercury content ofthe soil.

The present invention also protects the application of the primer-probeset in comparing the total mercury content of the soil in differentplots.

The present invention also protects a kit, which includes saidprimer-probe set.

The function of the kit is as the following (a) or (b):

(a) Detecting or assisting in detecting the total mercury content of thesoil;(b) Comparing the total mercury content of the soil in different plots.The kit also includes a carrier recording the Method A or the Method B.

Any of the above-mentioned soil is green land soil.

Any of the above-mentioned plots is a green land plot.

Any of the above-mentioned soil is green land soil in China.

Any of the above-mentioned plots is a green land plot in China.

Any of the above-mentioned soil is urban green land soil.

Any of the above-mentioned plots is an urban green land plot.

Any of the above-mentioned soil is green land soil in Yangtze RiverDelta of China.

Any of the above-mentioned plots is a green land plot in Yangtze RiverDelta of China.

Any of the above-mentioned soil is urban green land soil in YangtzeRiver Delta of China.

Any of the above-mentioned plots is an urban green land plot in YangtzeRiver Delta of China.

Any of the above-mentioned soil is park green land soil.

Any of the above-mentioned plots is a park green land plot.

Any of the above-mentioned soil is urban park green land soil.

Any of the above-mentioned plots is a urban park green land plot.

Any of the above-mentioned soil is park green land soil in Yangtze RiverDelta of China.

Any of the above-mentioned plots is a park green land plot in YangtzeRiver Delta of China.

Any of the above-mentioned soil is urban park green land soil in YangtzeRiver Delta of China.

Any of the above-mentioned plots is a urban park green land plot inYangtze River Delta of China.

Any of the above-mentioned soil is relocation site soil.

Any of the above-mentioned plots is a relocation site plot.

Any of the above-mentioned soil is relocation site soil in China.

Any of the above-mentioned plots is a relocation site plot in China.

Any of the above-mentioned soil is urban relocation site soil.

Any of the above-mentioned plots is an urban relocation site plot.

Any of the above-mentioned soil is relocation site soil in the YangtzeRiver Delta of China.

Any of the above-mentioned plots is a relocation site plot in theYangtze River Delta of China.

Any of the above-mentioned soil is urban relocation site soil in theYangtze River Delta of China.

Any of the above-mentioned plots is an urban relocation site plot in theYangtze River Delta of China.

The Yangtze River Delta refers to China's Shanghai, Jiangsu Province,and Zhejiang Province.

Any of the above-mentioned soil samples are taken from 0-20 cm surfacesoil.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the linear relationship between the copy number content ofthe target OTU and the total mercury content of the soil.

EMBODIMENTS

The following examples facilitate a better understanding of the presentinvention but do not limit the present invention. The experimentalmethods in the following examples, unless otherwise specified, are allconventional. The experimental materials used in the following examples,unless otherwise specified, are all purchased from conventionalbiochemical reagent stores. The quantitative experiments in thefollowing examples are all set to repeat the experiment three times, andthe results are averaged.

The soil quality indicators are determined according to relevantnational standards, industry standards and local standards, see Table 1for details.

TABLE 1 Determination methods of soil quality indicators DetectedIndicator Determination method pH LY/T 1239-1999 Determination of pHvalue in forest soil conductivity LY/T 1251-1999 Analysis methods ofwater soluble salts of forest soil ( conductivity method ) organic NY/T1121.6-2006 Method for determination matter of soil organic matter totalnitrogen LY/T 1228-2015 Nitrogen determination methods of forest soil(Kjeldahl method) available LY/T 1228-2015 Nitrogen determinationmethods nitrogen of forest soil (alkali-diffusion method) total LY/T1232-2015 Phosphorus determination phosphorus methods of forest soil(alkali fusion Mo-Sb anti spectrophotometric method) total LY/T1234-2015 Potassium determination potassium methods of forest soil(alkali fusion method) available DB31/T 661-2012 Appendix F AB-DTPAextraction/ phosphorus inductively coupled plasma mass spectrometeravailable DB31/T 661-2012 Appendix F AB-DTPA extraction/ potassiuminductively coupled plasma emission spectrometer available DB31/T661-2012 Appendix F AB-DTPA extraction/ sulfur inductively coupledplasma mass spectrometer available Refer to Appendix E (water saturatedextraction) chlorine in DB31/T 661-2012 exchangeable Refer to Appendix E(water saturated extraction) sodium in DB31/T 661-2012 total arsenicGB/T22105.2-2008 Soil quality-Analysis of total mercury, arsenic andlead contents- Atomic fluorescence spectrometry total bronze Totaldigestion inductively coupled plasma mass spectrometer method total zincTotal digestion inductively coupled plasma mass spectrometer methodtotal lead Total digestion inductively coupled plasma mass spectrometermethod total Total digestion inductively coupled plasma chromium massspectrometer method total nickel Total digestion inductively coupledplasma mass spectrometer method available Refer to DB31/T 661-2012Appendix F calcium AB-DTPA extraction/inductively coupled plasmaemission spectrometer available DB31/T 661-2012 Appendix F AB-DTPAextraction/ manganese inductively coupled plasma emission spectrometeravailable zinc DB31/T 661-2012 Appendix F AB-DTPA extraction/inductively coupled plasma emission spectrometer total mercury USEPA7473-2007 Thermal decomposition of atomic absorption spectrophotometry

Note: Compared with the reference method, the only difference inavailable calcium detection is that the detection target is availablecalcium.

Example 1. Discovery of OUT Related to Total Mercury Content in Soil

1. Collection of Soil Samples

In November 2017, research sample plots were set up in representativeparks and green lands in 16 administrative regions of Shanghai, China.

Sampling method: The collection of soil samples followed the principleof multi-point mixing. Choose 8 sampling points in each plot, use a 2.5cm diameter soil drill to collect 0-20 cm surface soil, and then mixthem into one soil sample.

A total of 76 soil samples were collected.

The soil samples were mixed evenly and passed through a 2 mm sieve toremove plant roots, gravel, and other debris. Then each soil sample wasdivided into two partitions. One partition was air-dried naturally, andthen as the sample for the determination of soil chemical properties instep 2; the other partition was stored at −80° C., and then as thesample used for the extraction of total soil DNA in step 3.

2. Analysis and Determination of Soil Quality Indicators

In step 1, the natural air-dried soil samples were analyzed anddetermined for soil quality indicators. The measurement results of soilquality indicators were shown in Table 2.

TABLE 2 Test results of soil quality indicators minimum maximal averageDetection Indicator value value value pH 5.32 8.79 7.92conductivity(μS/cm) 60.70 656.52 142.57 organic matter(g/kg) 7.10 46.6227.59 total nitrogen(g/kg) 0.47 2.34 1.13 available nitrogen(mg/kg)25.85 152.64 84.68 total phosphorus(g/kg) 0.42 1.00 0.68 totalpotassium(g/kg) 15.80 25.43 19.08 available phosphorus(mg/kg) 0.85 48.208.66 available potassium(mg/kg) 28.20 397.89 188.90 availablesulfur(mg/kg) 13.06 96.65 52.36 available chlorine(mg/L) 4.16 1900.0041.46 exchangeable sodium(mg/L) 3.30 993.00 21.60 total arsenic(mg/kg)4.81 13.50 8.73 total bronze(mg/kg) 16.38 99.84 36.05 total zinc(mg/kg)87.38 223.87 125.52 total lead(mg/kg) 18.24 52.18 29.01 totalchromium(mg/kg) 55.40 101.00 72.90 total nickel(mg/kg) 27.40 44.59 36.62available calcium(mg/kg) 198.36 366.95 282.89 available manganese(mg/kg)8.55 29.46 16.76 available zinc(mg/kg) 1.81 29.58 9.05 Totalmercury(mg/kg) 0.05 0.57 0.21

3. Diversity Analysis of Soil Archaea Population

(1). Extraction of Total DNA from Soil Samples Stored at −80° C. in Step1.

Total DNA of soil samples was extracted with MoBioPowerSoil® DNAIsolation Kit (MoBio Laboratories, Carlsbad, Inc., CA, USA). Each soilsample was extracted twice, and the total DNA extracted twice was mixedto obtain a DNA sample. For 76 soil samples, corresponding 76 DNAsamples were obtained. All DNA samples were stored at −80° C.

The DNA quality was examined by Nanodrop 2000 ultra-microspectrophotometer and 0.8% agarose gel electrophoresis (5V cm′, 45 min).The OD260/OD280 of the 76 DNA samples were in the range of 1.8-2.0, themaximum value of OD260/OD280 was 1.98, and the minimum value ofOD260/OD280 was 1.81.

(2). Archaea 16S rRNA Gene Amplification and High-Throughput Sequencing

The DNA sample was used as a template, and a primer pair composed ofprimer 524F-10-ext and primer Arch958R-mod was used for PCRamplification. After PCR amplification was completed, the PCR productswere subjected to 2% agarose gel electrophoresis, and then the targetbands were cut and purified by the GeneJET Gel Recovery Kit (ThermoScientific), and then the sequencing library was constructed, and theIllumina MiSeq sequencing platform (Illumina, San Diego, Calif., USA)was used for sequencing.

Primer 524F-10-ext and primer Arch958R-mod are universal primers forarchaea, and the target sequence is located in the V4-V5 variable regionof the archaea 16S rRNA gene.

524F-10-ext (SEQ ID NO. 4 of sequence listing):5′-TGYCAGCCGCCGCGGTAA-3′;Arch958R-mod (SEQ ID NO. 5 of sequence listing):5′YCCGGCGTTGAVTCCAATT-3′;

Y stands for C or T; V stands for G, A or C.

The reaction system for PCR amplification was 30 μL. The activeingredients were 15 μL, of Phusion® High-Fidelity PCR Master Mix (NewEngland Biolabs), primers and template DNA. In the reaction system, theconcentration of primer 524F-10-ext and primer Arch958R-mod were both0.2 μM. In the reaction system, the content of template DNA was 10 ng.

The PCR amplification reaction program: 95° C. pre-denaturation 3 min;95° C. denaturation 30 s, 55° C. annealing 30 s, 72° C. extension 45 s,33 cycles; 72° C. extension 5 min

(3). High-Throughput Data Analysis and Results

The specific steps for bioinformatics analysis of high-throughputsequencing results were as follows: 1) Extracted the same samplesequence from the original data according to the sample-specific labelto form a separate file, and removed the label and primer sequences; 2)FLASH (V1.2.7) software was used for sequence splicing; 3) Qiime(V1.7.0) software was used to perform quality filtering on the originalsequence after sequencing; 4) UCHIME software was used to detectchimeras and deleted them; 5) Uparse (v7.0.1001) software was used todivide Operational Taxonomic Units (OTUs) at a similarity level of 95%;6) Since the reliability of the single-copy sequence was questioned, thesingle-copy sequence was removed in the subsequent analysis; 7) In orderto remove the influence of different sequencing depths between samples,the sample OTU table was homogenized to the same sequencing depth; 8)Aligned the sequence of OTUs based on the RDP database and determinedthe taxonomic status thereof.

Total 27765 archaea 16S rRNA gene sequences were selected from eachsample for subsequent analysis. The archaea 16S rRNA gene sequences wereclassified at 95% sequence similarity, and a total of 580 OTUs wereobtained.

4. Correlation Analysis Between Soil Archaea Groups and Soil QualityIndicators

Performed Pearson correlation analysis on the 580 OTUs obtained in step3 and the soil chemical indicators data obtained in step 2.

The results show that among 580 OTUs, the abundance of archaeal OTU69 inthe soil has the strongest correlation with the total mercury content inthe soil; the correlation between the abundance of archaea OTU384 in thesoil and the total mercury content of the soil comes second; meanwhilethe abundance of archaea OTU69 in the soil was significantly negativelycorrelated with the total mercury content of the soil, with acorrelation coefficient r of −0.557; the correlation coefficient betweenthe abundance of archaea OTU384 in the soil and the total mercurycontent of the soil was −0.236. The abundance of archaea OTU69 orarchaea OTU384 in the soil can be used to reflect the total mercurycontent of the soil.

Example 2: Establishing a Linear Relationship Between OTU and SoilChemical Characteristics

1. Archaea Marker Gene Probe Design

According to the sequencing results, a probe for detecting OTU69 wasdesigned.

Probe69-1 probe(SEQ ID NO. 1 of sequence listing):5′-ACCCGCTCAACGGTTGGGCT-3′.Probe69-2 probe(SEQ ID NO. 6 of sequence listing):5′-TGATGGGATGGCCTCGAGCT-3′.

The Probe69-1 was a Taqman probe with a fluorescent group FAM at its5′end and a fluorescence quenching group TAMRA at its 3′end. TheProbe69-2 was a Taqman probe with a fluorescent group FAM at its 5′endand a fluorescence quenching group TAMRA at its 3′end.

According to the sequencing results, a probe for detecting OTU384 wasdesigned.

Probe384-1 probe(SEQ ID NO. 7 of sequence listing):5′-TGAACAGGCTTAGTGCCTATT-3′.Probe384-2 probe(SEQ ID NO. 8 of sequence listing):5′-AGTGCCTATTCAGTGCCGCA-3′.

The Probe384-1 was a Taqman probe with a fluorescent group FAM at its5′end and a fluorescence quenching group TAMRA at its 3′end. TheProbe384-2 was a Taqman probe with a fluorescent group FAM at its 5′endand a fluorescence quenching group TAMRA at its 3′end.

2. Determination of the Copy Number of Archaea Marker Gene in Soil

According to the measured value, soil samples with a gradientdistribution of total mercury content were randomly selected from thesoil samples in step 1 of Example 1.

(1) Took soil samples for extraction of the total DNA.

The extraction method of the total DNA was the same as that of step 3 ofExample 1.

(2) Took the template solution (that was, the total DNA solutionobtained in step 1), and performed fluorescent quantitative PCR (probemethod) on the LightCycler® 96 real-time fluorescent quantitative PCRinstrument.

A primer pair consisting of the primer 524F-10-ext and the primerArch958R-mod was used. Probe69-1 probe or Probe69-2 probe or Probe384-1probe or Probe384-2 probe was used.

Reaction system (20 μL): 10 μL, Premix Ex Taq (Takara, Dalian, China),0.4 μL, primer 524F-10-ext, 0.4 μL, primer Arch958R-mod, 0.2 μL, probe,2 μL template solution and 7 μL sterile water. In the reaction system,the concentration of primer 524F-10-ext was 0.2 μM, the concentration ofprimer Arch958R-mod was 0.2 μM, and the concentration of probe was 0.1μM. In the reaction system, the content of template DNA was Ing.

Reaction program: 95° C. pre-denaturation for 120 s; 95° C. denaturationfor 10 s, 60° C. annealing extension for 45 s, 45 cycles.

The specificity of amplification was determined by the melting curve.

The Ct value was substituted into the standard curve equation to obtainthe copy number of the target OTU, and then the copy number content(unit was copy number/g, that was, the copy number of the target OTUineach gram of the dry soil sample) of the target OTU in the soil samplewas calculated.

The preparation method of the standard curve equation was shown in step3.

(3) Constructed a standard curve for real-time PCR

Ligated the DNA molecule (amplified from total soil DNA) shown as SEQ IDNO.2 of the sequence listing to the pMD18-T vector to obtain OTU69standard plasmid. Using TE buffer as the solvent, prepared the standardplasmid solutions containing different concentrations of OTU69 standardplasmid (the DNA concentration in the standard plasmid solution wasmeasured by Nanodrop 2000 ultra-micro spectrophotometer, and wasconverted into DNA copy number, which was the OTU69 copy number). Eachstandard plasmid solution was used as a template solution, and detectionwas performed according to the method of step 2 (using the Probe69-1probe or the Probe69-2 probe) to obtain the standard curve equation withthe logarithm of OTU69 copy number (logarithm with base 10) as theindependent variable and Ct as the dependent variable.

SEQ ID NO. 2: TGTCAGCCGCCGCGGTAATACCAGCACCCCGAGTGGTCGGGACGATTATTGGGCCTAAAGCATCCGTAGCCGGTCATGCAAGTCTTCCGTTAAATCCACCCGCTCAACGGTTGGGCTGCGGAGGATACTACGTGGCTAGGAGGCGGGAGAGGCAAGCGGTACTCAGTGGGTAGGGGTAAAATCCTTTGATCCATTGAAGACCACCAGTGGCGAAGGCGGCTTGCCAGAACGCGCTCGACGGTGAGGGATGAAAGCTGGGGGAGCAAACCGGATTAGATACCCGGGTAGTCCCAGCTGTAAACGATGCAGACTCGGTGATGGGATGGCCTCGAGCTATCCCAGTGCCGCAGGGAAGCCGTTAAGTCTGCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTTAAAGGAATTGGCGGGGGAGCACCACAAGGGGTGAAGCCTGCGGTTCAATTGGATTCAA CGCCGGA

Ligated the DNA molecule (amplified from total soil DNA) shown as SEQ IDNO.3 of the sequence listing to the pMD18-T vector to obtain OTU384standard plasmid. Using TE buffer as the solvent, prepared standardplasmid solutions containing different concentrations of OTU384 standardplasmid (the DNA concentration in the standard plasmid solution wasmeasured by Nanodrop 2000 ultra-micro spectrophotometer, and wasconverted into DNA copy number, which was the OTU384 copy number). Eachstandard plasmid solution was used as a template solution, and detectionwas performed according to the method of step 2 (using the Probe384-1probe or the Probe384-2 probe) to obtain the standard curve equationwith the logarithm of OTU384 copy number (logarithm with base 10) as theindependent variable and Ct as the dependent variable.

SEQ ID NO. 3: TGTCAGCCGCCGCGGTAATACCAGCACCCCGAGTGGTCGGGACGATTATTGGGCCTAAAGCATCCGTAGCCGGTTCTACAAGTCTTCCGTTAAATCCACCTGCTTAACAGATGGGCTGCAGAGGATACTATAGAGCTAGGAGGCGGGAGAGGCAAGCGGTACTTAGTGGGTAGGGGTAAAATCCGTTGATCCACTGAAGACCACCAGTGGCGAAGGCGGCTTGCCAGAACGCGCTCGACGGTGAGGGATGAAAGCTGGGGGAGCAAACCGGATTAGATACCCGGGTAGTCCCAGCTGTAAACGATGCAGACTCGGTGATGAACAGGCTTAGTGCCTATT CAGTGCCGCAGGGAAGCCGTTAAGTCTGCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTTAAAGGAATTGGCGGGGGAGCACCACAAGGGGTGAAGCCTGCGGTTCAATTGGATTCAA CGCCGGA

The efficiency of fluorescence quantitative PCR amplification wasbetween 90% and 110%.

3. Establishment of a Linear Relationship Between Copy Number of ArchaeaMarker Gene and Chemical Characteristics of the Soil

The results of the measured total mercury values (unit: mg/kg), Ctvalues, and target OTU copy number content (unit: copy number/g) of thesoil samples were shown in Table 3. The measured values of total mercuryof the soil samples were the data obtained in step 2 of Example 1. TheCt values and the copy number content of the target OTU of the soilsamples were the data obtained in step 2 of the present embodiment.Probe69-1 probe was used to perform fluorescence quantitative PCR, therelative abundance of target OTU of the soil (reflected as the copynumber of target OTU in the soil) had a good linear relationship withthe total mercury content of the soil.

TABLE 3 measured Probe69-1 Probe69-2 Probe384-1 Probe384-2 soil value ofcopy copy copy copy sample total number number number number numbermercury Ct value content Ct value content Ct value content Ct valuecontent 1 0.57 22.22 7.4 × 10⁶ 20.36 5.3 × 10⁶ 22.40 5.2 × 10⁷ 21.61 5.4× 10⁷ 2 0.46 21.76 1.0 × 10⁷ 20.40 5.1 × 10⁶ 22.70 4.2 × 10⁷ 22.04 3.9 ×10⁷ 3 0.39 21.38 1.3 × 10⁷ 19.81 7.8 × 10⁶ 22.92 3.6 × 10⁷ 22.15 3.6 ×10⁷ 4 0.26 20.69 2.0 × 10⁷ 19.87 7.5 × 10⁶ 22.76 4.0 × 10⁷ 21.90 4.3 ×10⁷ 5 0.15 20.35 2.5 × 10⁷ 19.78 8.0 × 10⁶ 22.37 5.3 × 10⁷ 21.67 5.2 ×10⁷ 6 0.06 19.86 3.5 × 10⁷ 19.48 1.0 × 10⁷ 21.58 9.1 × 10⁷ 20.56 1.2 ×10⁸

Note: soil sample 1, collected from a green land of the park inSongjiang District; soil sample 2, collected from a green land of thepark in Jinshan District; soil sample 3, collected from a green land ofthe park in Huangpu District; soil sample 4, collected from a green landof the park in Jing'an District; soil sample 5, collected from a greenland of the park in Putuo District; soil sample 6, collected from agreen land of the park in Minhang District.

Probe69-1 probe was used to perform fluorescence quantitative PCR, thelinear relationship between the copy number content of the target OTU inthe soil and the total mercury content of the soil was shown in FIG. 1.The linear equation is y=−0.1811x+0.6508; R²=0.959; y represents thetotal mercury content (mg/kg), and x represents the copy number contentof the target OUT (×10⁷ copies/g).

Example 3. Molecular Detection of Total Mercury Content of Soil Samplesof Unknown Urban Green Lands

Randomly selected three green lands of urban parks in Shanghai, Nanjingand Suzhou in China.

Sampling method: The collection of soil samples followed the principleof multi-point mixing: selected 8 sampling points in each plot,collected 0-20 cm surface soil using a 2.5 cm diameter soil drill, andthen mix the collected soil into one soil sample.

The soil samples were mixed evenly and passed through a 2 mm sieve toremove plant roots, gravel, and other debris. Then each soil sample wasdivided into two parts. One part was air-dried naturally, and then as asample to be detected to get the actual measured value of the totalmercury content (unit: mg/kg). The other part was stored at −80° C. andthen as a sample for extraction of total soil DNA.

1. Extraction of total DNA of soil samples. Adopted MoBioPowerSoil® DNAIsolation Kit (MoBio Laboratories, Carlsbad, Inc., CA, USA). Extractedeach soil sample two times and mix all of the extracted total DNA to geta DNA sample.

2. Took the template solution (i.e. the DNA sample obtained in step 1),and performed fluorescent quantitative PCR (probe method) on theLightCycler® 96 real-time fluorescent quantitative PCR instrument.

Reaction system (20 μL): 10 μL, Premix Ex Taq (Takara, Dalian, China),0.4 μL, primer 524F-10-ext, 0.4 μL, primer Arch958R-mod, 0.2 μL,Probe69-1 probe, 2 μL, template solution and 7 μL, sterile water. In thereaction system, the concentration of primer 524F-10-ext was 0.2 μM, theconcentration of primer Arch958R-mod was 0.2 μM, and the concentrationof Probe69-1 probe was 0.1 μM. In the reaction system, the content oftemplate DNA was Ing.

Reaction program: 95° C. pre-denaturation for 120 s; 95° C. denaturationfor 10 s, 60° C. annealing extension for 45 s, 45 cycles.

The Ct value was substituted into the standard curve equation to obtainthe copy number of OTU69, and then the copy number content of OTU69 ofthe soil sample was calculated (the unit was copy number/g, that was,the number of copies of OTU69 per gram of dry weight of soil sample).

The construction method of the standard curve equation was shown in step(3) of part 2 of Example 2.

3. The copy number content of OTU69 of the soil sample was substitutedinto the linear equation to obtain the calculated value of the totalmercury content of the soil sample (unit: mg/kg).

The linear equation is: y=−0.1811x+0.6508; R2=0.959; wherein yrepresents the total mercury content (mg/kg), x represents the copynumber content of OTU69 (×10⁷ copies/g).

The results of the copy number content of OTU69 in the soil sample(unit: copy number/g), the calculated value of the total mercury contentof the soil sample (unit: mg/kg), and the measured value of the totalmercury content of the soil sample (unit: mg/kg) are shown in Table 4.

TABLE 4 The copy The calculated The measured number value value ofcontent of of the total the total OTU69 mercury mercury (copy contentcontent number/g) ( mg/kg ) ( mg/kg ) Soil sample 1 (Shanghai) 1.9 × 10⁷0.31 0.22 Soil sample 2 (Shanghai) 3.2 × 10⁷ 0.07 0.09 Soil sample 3(Shanghai) 2.8 × 10⁷ 0.14 0.15 Soil sample 1 (Nanjing) 8.2 × 10⁶ 0.500.46 Soil sample 2 (Nanjing) 3.0 × 10⁷ 0.10 0.12 Soil sample 3 (Nanjing)3.2 × 10⁷ 0.07 0.09 Soil sample 1 (Suzhou) 1.6 × 10⁷ 0.37 0.27 Soilsample 2 (Suzhou) 2.0 × 10⁷ 0.29 0.19 Soil sample 3 (Suzhou) 3.4 × 10⁷0.04 0.06

Example 4. Molecular Detection of the Total Mercury Content of the SoilSamples from Unknown Relocation Sites of Cities

In Shanghai, Nanjing, and Suzhou in China, three city relocation siteswere randomly selected.

The method was the same as in Example 3.

The results of the copy number content of OTU69 in the soil sample(unit: copy number/g), the calculated value of the total mercury contentof the soil sample (unit: mg/kg), and the measured value of the totalmercury content of the soil sample (unit: mg/kg) are shown in Table 5.

TABLE 5 The copy The calculated The measured number value value ofcontent of the the total of OTU69 total mercury mercury (copy contentcontent number/g) ( mg/kg ) ( mg/kg ) Soil sample 1 (Shanghai) 6.3 × 10⁶0.54 0.69 Soil sample 2 (Shanghai) 1.1 × 10⁷ 0.45 0.31 Soil sample 3(Shanghai) 2.4 × 10⁷ 0.22 0.16 Soil sample 1 (Nanjing) 2.0 × 10⁷ 0.290.19 Soil sample 2 (Nanjing) 1.4 × 10⁷ 0.40 0.29 Soil sample 3 (Nanjing)2.4 × 10⁷ 0.22 0.16 Soil sample 1 (Suzhou) 2.9 × 10⁷ 0.13 0.14 Soilsample 2 (Suzhou) 7.4 × 10⁶ 0.52 0.66 Soil sample 3 (Suzhou) 1.3 × 10⁷0.42 0.30

INDUSTRIAL APPLICATION

The invention discloses a method for detecting the total mercury contentof the soil or comparing the total mercury content of the soil betweendifferent plots, which has the following advantages: detecting the totalmercury content of the soil or comparing the total mercury content ofthe soil between different plots, and small sample demand, no need forpre-treatment, short required time, low labor cost, realizing the rapidautomatic detection of large quantities of samples. The presentinvention deserves to apply and promote in the evaluation of soilsamples.

1. A method for detecting the total mercury content in the soil,includes the following steps: using the total DNA of the soil sample asa template to perform real-time fluorescent quantitative PCR; theamplification primer pair of real-time fluorescence quantitative PCR iscomposed of primer 524F-10-ext shown as SEQ ID NO.4 of the sequencelisting and primer Arch958R-mod shown as SEQ ID NO.5 of the sequencelisting; the nucleotide sequence of the probe for real-time fluorescencequantitative PCR is shown as SEQ ID NO.1 of the sequence listing;obtaining the Ct value by real-time fluorescent quantitative PCR andcalculating the copy number according to the Ct value, and calculatingthe total mercury content by the copy number content in the soil sample.2. The method of claim 1, wherein the method further comprising: testingthe soil samples of more than two plots, comparing the total mercurycontent in the soil of each plot based on the test results.
 3. A DNAmolecule, as shown in SEQ ID NO.1 of the sequence listing. 4-8.(canceled)
 9. A kit comprising primer-probe set, which is composed of aspecific primer pair and a specific probe; wherein the specific primerpair is composed of primer 524F-10-ext shown in SEQ ID NO.4 of thesequence listing and primer Arch958R-mod shown in SEQ ID NO.5 of thesequence listing; the nucleotide sequence of the specific probe is shownin SEQ ID NO.1 of the sequence listing. 10-12. (canceled)